A major focus of the Waldmann Laboratory is the definition of the molecular abnormalities of T-cell malignancy. Using RNA sequencing on purified leukemic cells of 27 patients with ATL and 11 ATL cell lines from patients' paired cells that remain in the germline configuration we demonstrated a number of molecular abnormalities. Five patient ATL cells/lines manifested NOTCH1 mutations. There were six CARD11 missense mutations. Furthermore there were four nonsense mutations of CCR4 observed in 8 ATL samples (18%). CCR4 is a chemokine receptor highly expressed in ATL cells and in normal TH17 and T reg cells. It is the target of our new clinical trial in patients with ATL using the KW-0761 monoclonal antibody, mogamulizumab. Nine recurrent somatic mutations of CCR4 encoding CCR were detected in 14/53 ATL samples (26%) that consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 and Y331 in the carboxyl terminal. Functionally the CCR4-Q330, a nonsense isoform, was a gain of function since it increased cell migration towards the CC4R ligands, CCL17 and CCL22 in part by impairing receptor internalization. This mutant enhanced PI (3) kinase/AKT activation following receptor engagement by CCL22 in ATL cells and conferred a relative advantage in long-term in vitro cultures. These findings implicate somatic gain of function CCR mutations in the pathogenesis of ATL. A dominant element in our efforts to define the cellular abnormalities in ATL was focused on the disordered c cytokine receptor (IL-2, IL-2R, IL-15, IL-15R). We demonstrated previously that HTLV-1 Tax transactivates 2 autocrine (IL-2/IL-2R/IL-15/IL-15R) and a paracrine loop that involves IL-9 production by ATL cells. To extend these observations, we performed molecular interference Achilles' heel screening of 14 ATL cell lines employing a library of retroviral vectors for inducible expression of short hairpin RNAs (shRNAs) to identify genes essential for leukemic cell survival. Using this process of loss-of-function screen, 6 of 7 distinct cytokine dependent ATL cell lines were shown to be critically dependent on JAK1 and JAK3 for proliferation and survival. The critical nature of the IL-2, IL-9, IL-15, JAK1 and JAK3 pathway for ATL was supported by our observation that the ex vivo 6-day culture of PBMCs from patients with smoldering and chronic ATL could be inhibited by the administration of antibodies to the IL-2 receptor, IL-15 and IL-9. Furthermore, we demonstrated that the pan JAK inhibitor, tofacitinib and the JAK1/2 inhibitor, ruxolitinib inhibited the ex vivo proliferation of PBMCs from ATL patients by over 60%. On the basis of these insights showing the critical nature of the gamma cytokine JAK/STAT system in ATL we have initiated a clinical trial involving the JAK1/2 inhibitor, ruxolitinib in patients with ATL. To extend these studies we are searching for novel multicomponent/combination drug therapies for ATL by applying high-throughput matrix screening for cellular signaling on ATL cell lines to define combination therapies that identify agents with synergy. Optimal synergy was demonstrated between the JAK1/2 inhibitor, ruxolitinib added in association with the Bcl-xL inhibitor, navitoclax. Examination of the mechanistic underpinnings of this combination highlighted a stimulation Bim and Puma expression and reduced phosphorylation of Bad upon cellular exposure to ruxolitinib. The combination was further noted to strongly activate BAX and effect mitochondrial depolarization that led to PARP and Mcl-1 cleavage from a 40 kDa anti-apoptotic to a 24 kDa pro-apoptotic form. Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, while the combination dramatically lowered tumor burden and prolonged survival in an aggressive ATL murine model. Critically this drug combination strongly blocked proliferation of ex vivo cultures of 5 ATL patients' PBMCs. These studies provide support for a therapeutic trial in patients with smoldering and chronic ATL using a combination of agents that inhibit JAK1/3 and the anti-apoptotic Bcl-2 family members. In contrast to cytokine dependent cell lines in smoldering and chronic ATL, cytokine independent ATL cell lines in patients with acute ATL do not respond to JAK inhibitors. To develop an alternative therapeutic strategy we took advantage of the high expression of the CC chemokine receptor-4 (CCR4)--a hallmark of ATL present in over 90% of cases. Clinical trials in ATL of the therapeutic monoclonal antibody mogamulizumab directed against CCR4 are ongoing and promising. Although a 50% response rate was observed with anti-CCR4 in ATL the antibody is not curative and leukemic relapses are the rule. In an effort to define agents that provide additive/synergy when used in combination with anti-CCR4 we applied matrix screening for cellular signaling on 5 ATL cell lines to define active agents. We chose two agents that provide epigenetic modifications of ATL, romidepsin and 5-azacytidine. We investigated the therapeutic efficacy of these agents in a murine model of human ATL, the 43Tb- model. Each of the three agents when added alone inhibited tumor growth as monitored by tumor size and prolonged survival of leukemia-bearing mice compared with that of the control group. The combination of three agents significantly enhanced antitumor efficacy as assessed by tumor size, surrogate tumor markers in the serum (human IL-2R alpha) and survival of 43Tb- tumor-bearing mice compared with all other treatment groups. The improved efficacy observed by combining romidepsin, 5-azacytidine and anti-CCR4 supports a clinical trial to evaluate this combination in the treatment of acute ATL.